8EVF

HUMAN DNA POLYMERASE ETA EXTENSION COMPLEX WITH AN INCOMING DCTP

Summary for 8EVF

• Entry DOI: 10.2210/pdb8evf/pdb
• Descriptor: DNA polymerase eta, DNA 5'-D(P*TP*GP*(MO1)P*TP*GP*AP*CP*GP*CP*T-3', DNA (5'-D(*AP*GP*CP*GP*TP*CP*AP*G)-3'), ... (6 entities in total)
• Functional Keywords: dna damage, dna polymerase, lesion bypass, y-family polymerase, translesion dna synthesis (tls), dna binding protein, transferase-dna complex, replication, transferase/dna
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 3
• Total formula weight: 55347.08

Authors

Pallan, P.S.,Egli, M. (deposition date: 2022-10-20, release date: 2023-08-02, Last modification date: 2023-08-30)

Primary citation

Richie-Jannetta, R.,Pallan, P.,Kingsley, P.J.,Kamdar, N.,Egli, M.,Marnett, L.J.
The peroxidation-derived DNA adduct, 6-oxo-M 1 dG, is a strong block to replication by human DNA polymerase eta.
J.Biol.Chem., 299:105067-105067, 2023

PubMed Abstract: The DNA adduct 6-oxo-MdG, (3-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-oxo-pyrimido(1,2alpha)purin-10(3H)-one) is formed in the genome via oxidation of the peroxidation-derived adduct MdG. However, the effect of 6-oxo-MdG adducts on subsequent DNA replication is unclear. Here we investigated the ability of the human Y-family polymerase hPol η to bypass 6-oxo-MdG. Using steady-state kinetics and analysis of DNA extension products by liquid chromatography-tandem mass spectrometry, we found hPol η preferentially inserts a dAMP or dGMP nucleotide into primer-templates across from the 6-oxo-MdG adduct, with dGMP being slightly preferred. We also show primer-templates with a 3'-terminal dGMP or dAMP across from 6-oxo-MdG were extended to a greater degree than primers with a dCMP or dTMP across from the adduct. In addition, we explored the structural basis for bypass of 6-oxo-MdG by hPol η using X-ray crystallography of both an insertion-stage and an extension-stage complex. In the insertion-stage complex, we observed that the incoming dCTP opposite 6-oxo-MdG, although present during crystallization, was not present in the active site. We found the adduct does not interact with residues in the hPol η active site but rather forms stacking interactions with the base pair immediately 3' to the adduct. In the extension-stage complex, we observed the 3' hydroxyl group of the primer strand dGMP across from 6-oxo-MdG is not positioned correctly to form a phosphodiester bond with the incoming dCTP. Taken together, these results indicate 6-oxo-MdG forms a strong block to DNA replication by hPol η and provide a structural basis for its blocking ability.

PubMed: 37468099
DOI: 10.1016/j.jbc.2023.105067

Experimental method

X-RAY DIFFRACTION (2.87 Å)